DESCRIPTION: This proposal addresses the clinical need for more effective therapies to promote repair of chronic wounds. Deficits in vascular supply can lead to chronic ulceration and increase susceptibility to infection and ultimately to limb amputation. Biotherapies represent a promising class of drugs for jump starting endogenous wound repair processes. However, current clinical products such as platelet derived growth factor (PDGF) have only a modest impact on complete healing. We propose to engineer a transformative therapeutic approach for effective and safe delivery of therapeutic small interfering ribonucleic acids (siRNA) to impaired skin wounds. The proposed siRNA post- transcriptionally blocks translation of prolyl hydroxylase domain 2 (PHD2), an enzyme that negatively regulates stability of hypoxia inducible factor 1 alpha (HIF1?). PHD2 siRNA, as a result, stabilizes HIF1? and activates transcription of a host of related genes that exert positive effects on vascular growth, vessel maturation/stabilization, and stem cell recruitment. A recent publication from our team supports this concept, and we propose that translational therapeutic approaches for activation of HIF1? will stimulate more robust healing relative to current approaches that use a single growth factor with narrow bioactivity. Existing technologies for therapeutic delivery of siRNA consist of cationic lipids or polymers that are formulated into nanocarriers, an approach that can evoke nonspecific inflammatory effects. These collateral effects can limit translation into clinical use. The overall goal of this proposal is to engineer a clinically translatable (simple, effective, and safe), nanocarrier-free delivery system for substrate mediated delivery of PHD2 siRNA from a fully cell-degradable biomaterial that promotes tissue vascularization and repair. The proposed biomaterial will serve as both a cell-inductive, porous tissue template and a depot for sustained, controlled delivery of siRNA at the cell- biomaterial interface. The first aim of the proposal is t synthesize siRNA conjugates for nanoparticle-free, scaffold- mediated delivery of siRNA and to test their effectiveness in vitro. The second aim is to characterize and optimize substrate-mediated siRNA delivery systems in vivo using mouse subcutaneous implant and diabetic rat excisional wound models. In addition to in vivo validation of our delivery platform, another key aspect of this aim is to explore the impact of the chemistry of the scaffolding itself (i.e., hydrolytically degradable versus cell degradable) on the effectiveness of substrate mediated PHD2 siRNA delivery and on wound outcomes. The third aim is to test our success at promoting vascularization and healing in the setting of highly compromised, ischemic wounds. We will first confirm bioactivity and optimize our delivery system in rats and then proceed to studies in pigs, which have thicker skin that better models human cutaneous anatomy and physiology. Successful completion of this aim in a well-accepted preclinical model will yield a technology poised for translation into a clinical wound therapy and will inform whether our therapeutic is most appropriate for diabetic and/or purely ischemic wound settings. Our interdisciplinary team includes two bioengineers, a pathologist, and a skin wound scientist and is well-positioned to design and translate innovative drug and device technologies for wound therapy.